DNA Structural Elements Required for ERCC1-XPF Endonuclease Activity

Abstract

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5 incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn 2؉ concentrations (0.2 mM). A minimum of 4 -8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3 protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5 side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3 or 5 direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination. Nucleotide excision repair (NER)1 guards the integrity of the genome by removing bulky adducts and the most prominent UV-induced lesions from the DNA. During NER, the damaged DNA strand is incised asymmetrically around the lesion to allow release of the damage as part of a 24 -32-nucleotide DNA fragment, followed by resynthesis and ligation (1-3). Whereas the latter steps are accomplished by general replication factors such as proliferating cell nuclear antigen, replication factor C, DNA polymerase ␦, and/or ⑀ and ligase, the incision stage of NER requires the action of XP factors (XPA-G) (4). XPA, and perhaps XPC complexed with hHR23B, are thought to be involved in damage recognition; XPB and XPD, two helicases present in the basal transcription factor complex TFIIH, and replication protein A (RPA) presumably demarcate the lesion by local opening of the DNA; XPG and the heterodimeric complex ERCC1-XPF perform the actual DNA cleavage. The role of XPE is, as yet, unclear. Mutations in either of the XP factors can cause the extreme UV-sensitive and cancer-prone phenotype observed with Xeroderma pigmentosum (XP) patients. Some human XP genes cross-complement the NER defect of laboratory induced UV-sensitive Chinese hamster mutant cell lines, subdivided into 10 complementation groups. Human XPF, for example, corrects the repair-defect of rodent group 4 cell lines. The human excision repair cross-complementing gene ERCC1 corrects rod...

RPA activates the XPF ‐ ERCC 1 endonuclease to initiate processing of DNA interstrand crosslinks

“…However, at the same XPF‐ERCC1 concentration, only ~5% of a fork substrate bearing a model nascent leading strand (5′‐flap structure, here denoted as “+leading‐strand” structure) is incised, in line with previous reports that Rad1‐Rad10 (the budding yeast homologues of XPF‐ERCC1) incisions are inhibited on a 5′‐flap structure (Rodriguez et al , 1996; Fig 1A–C, confirmed with 5′‐labelled substrates in Fig EV2). Moreover, on a fork structure containing a model nascent lagging strand (3′‐flap structure, denoted as “+lagging‐strand” structure) or model fork with both nascent leading and lagging strands, we did not detect XPF‐ERCC1 incisions, consistent with the reported inhibition of XPF‐ERCC1 on 3′‐flap structures versus simple fork structures (Rodriguez et al , 1996; de Laat et al , 1998a; Figs 1A and EV2). We also confirmed that as expected, only the strand with a 3′‐flap structure (the labelled strand in Figs 1A and EV2) is incised by XPF‐ERCC1, for all structures tested (Fig EV3A).…”

Multiple DNA Binding Domains Mediate the Function of the ERCC1-XPF Protein in Nucleotide Excision Repair

“…We titrated 3Ј-labeled splayed arm, stem loop or duplex DNA substrates with wild-type ERCC1-XPF and measured the increase in anisotropy as a function of the protein concentration. Surprisingly, ERCC1-XPF-bound dsDNA with similar affinity as the splayed arm or stem-loop substrates (data not shown), although ERCC1-XPF did not incise dsDNA (51). However, addition of a 10-fold excess of non-labeled dsDNA abolished the binding of ERCC1-XPF to dsDNA, but not to the splayed arm and stem loop substrates, confirming that ERCC1-XPF specifically bound ss/dsDNA junctions.…”

“…S1B). The endonuclease activity of ERCC1-XPF in wild-type and mutant form was tested on a 3Ј fluorescently labeled stem-loop model substrate, in which the protein cut at the ss and dsDNA junction in the presence of Mg 2ϩ (51). ERCC1-XPF in wild-type form and with mutations in the HhH domain of XPF or the central domain of ERCC1, cleaved the stem-loop substrate with similar efficiencies as the wild-type protein in a concentration-dependent manner (Fig.…”

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors

“…complex purification was performed as described previously (14). Briefly, Sf9 cells were infected with ERCC1 and XPF-His-tagged baculoviruses.…”

“…After being recognized by the XPC/HR23B complex, the damaged DNA structure is targeted by TFIIH, which recruits the other factors upon the addition of ATP (9 -11). The unwound DNA is then incised by the two endonucleases XPG and XPF/ERCC1 on the 3Ј and 5Ј side of the lesion, respectively (12)(13)(14)(15), leaving a gap structure that is filled up by the DNA polymerase ⑀ or ␦ and the accompanying factors PCNA, RF-C, RPA, and DNA ligase I (16). Whether or not the NER reaction occurs by sequential arrival of the various factors or by a pre-assembled complex referred to as the repairosome or the holoenzyme is still under debate (17)(18)(19).…”